Liposome compositions of porphyrin photosensitizers

ABSTRACT

Liposomal pharmaceutical formulations incorporating porphyrin photosensitizers useful for photodynamic therapy or diagnosis of malignant cells. The liposomal formulations comprise a porphyrin photosensitizer, particularly the hydro-mono benzoporphyrins (BPD) having light absorption maxima in the range of 670–780 nanometers, a disaccharide or polysaccharide and one or more phospholipids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is Continuation of U.S. Ser. No. 09/589,358 filed Jun.8, 2000 now U.S. Pat. No. 6,890,555, which is a Continuation of U.S.Ser. No. 08/489,850 now U.S. Pat. No. 6,074,666 filed Jun. 13, 1995,which is a Continuation of U.S. Ser. No. 07/832,542 filed Feb. 5, 1992,abandoned. The contents of these documents are incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates to improved pharmaceutical formulations comprisingliposomes incorporating porphyrin photosensitizers. Specifically, theinvention is directed to a freeze-dried pharmaceutical formulationcomprised of a porphyrin photosensitizer, a disaccharide orpolysaccharide and one or more phospholipids which, upon reconstitutionwith a suitable aqueous vehicle, forms liposomes containing theporphyrin photosensitizer. Particular porphyrin photosensitizers whichare advantageously employed in the practice of this invention includethe hydro-monobenzoporphyrins having a light absorption maxima in therange of 670–780 nanometers. The photosensitizing formulations areuseful to mediate the destruction of unwanted cells or tissues or otherundesirable materials by irradiation or to detect their presence throughflourescence.

DESCRIPTION OF THE RELATED ART

The use of porphyrin compounds, and in particular hematoporphyrin andhematoporphyrin derivative (HPD), have been known for some time to beuseful systemically when combined with light, for the treatment anddiagnosis of malignant cells. The porphyrins appear to naturally“localize” in malignant tissue where they absorb light at certainwavelengths when irradiated, providing a means to detect the tumor bythe location of the fluorescence. Accordingly, preparations containingthe porphyrins are useful in the diagnosis and detection of such tumortissues. (See, e.g. “Porphyrin Photosensitization”, Kessel, D., et al.,eds. (1983) Plenum Press). In addition, the porphyrins also have thecapability of exhibiting a cytotoxic effect on the cells or othertissues in which they are localized when exposed to light at theappropriate wavelength. (See, e.g., Diamond, I., et al., Lancet (1972)2: 1175–1177; Dougherty, T. J. et al., “The Science of Photo Medicine”(1982) J. D. Regan & J. A. Parrish, eds., pp. 625–638; Dougherty, T. J.,et al., “Cancer: Principles and Practice of Oncology” (1982) V. T.DeVita Jr., et al., eds., pp. 1836–1844). It has been postulated thatthe cytotoxic effect of the porphyrins is due to the formation ofsinglet oxygen when exposed to light (Weishaupt, K. R., et al., CancerResearch, (1976) 36: 2326–2329). The successful treatment ofAIDS-related oral Kaposi's Sarcoma with a purified form of HPD,Photofrin® porfimer sodium, was described in Schwietzer, V. G. et al.,Otolaryngology—Head and Neck Surgery (1990) 102: 639–649.

In addition to systemic use for the treatment and diagnosis of tumors,the porphyrins can be used in a variety of other therapeuticapplications. For example, photosensitizers are useful in the detectionand treatment of artherosclerotic plaques as disclosed in U.S. Pat. Nos.4,517,762 and 4,577,636. U.S. Pat. Nos. 4,500,507 and 4,485,806 describethe use of radiolabeled porphyrin compounds for tumor imaging. Porphyrincompounds have also been used topically to treat various skin diseasesas disclosed in U.S. Pat. No. 4,753,958.

A number of porphyrin photosensitizer preparations have been disclosedfor therapeutic applications. A photosensitizer preparation widely usedin the early stages of photodynamic therapy both for detection andtreatment was a crude derivative of hematoporphyrin, also calledhematoporphyrin derivative (HPD) or Lipson derivative, prepared asdescribed by Lipson et al., J. Natl. Cancer Inst. (1961) 26: 1–8. Apurified form of the active component(s) of HPD was prepared byDougherty and co-workers by adjustment of the pH to cause aggregationand recovery of the aggregate, as disclosed in U.S. Pat. Nos. 4,649,151,4,866,168, 4,889,129 and 4,932,934. A purified form of this product isused clinically under the trademark Photofrin® porfimer sodium. Ofparticular interest in the context of the present invention are a groupof modified porphyrins, known as “green porphyrins” (Gp), having a lightabsorption maximum between 670–780 nm which have been shown to confercytotoxicity to target cells at concentrations lower than those requiredfor hematoporphyrin or HPD. These Gp are obtained using Diels-Alderreactions of acetylene derivatives with protoporphyrin under appropriateconditions. The preferred forms of Gp are the hydro-monobenzoporphyrinderivatives (“BPD” ). The preparation and use of the Gp and BPDcompounds are disclosed in U.S. Pat. No. 4,920,143 and U.S. Pat. No.4,883,790, hereby incorporated by reference into the disclosure of thepresent application.

While the porphyrin compounds naturally have the ability to localize inneoplastic tissue while being cleared from the normal surroundingtissue, the selectivity of the porphyrin sensitizers is somewhatlimited. Because tumor tissues generally include various components suchas malignant cells, the vascular system, macrophages, fibroblasts, etc.,the distribution of the photosensitizer in the tissue may be highlyheterogeneous, especially for those photosensitizers which are nothomogeneous and contain a mixture of components with different degreesof hydro or lipo-solubility. Zhou, C. et al., Photochemistry andPhotobiology, (1988) 48: 487–492. The low selectivity of some of thesetumors as tumor localizers may lead to side effects such ashypersensitivity exhibited nonspecifically throughout the organism.Therefore, an active area of research is to increase the tumorselectivity of known porphyrin photosensitizers and to identify thoseporphyrin photosensitizers which exhibit higher tumor-selectivity. Ingeneral, those photosensitizers which are more lipophilic tend toexhibit greater tumor targeting. J. D. Spikes, et al., Lasers in MedicalScience, (1986) 2: 3, 3–15.

It has recently been shown that the encapsulation of certain drugs inliposomes before administration has a marked effect on thepharmoco-kinetics, tissue distribution, metabolism and efficacy of thetherapeutic agent. Liposomes are completely closed lipid bilayermembranes containing an entrapped aqueous volume which are formedspontaneously on addition of an aqueous solution to a dry lipid film.They may be unilamellar vesicles possessing a single membrane bilayer ormultilamellar vesicles having multiple membrane bilayers, each separatedfrom the next by an aqueous layer. The bilayer is composed of two lipidmonolayers having a hydrophobic “tail” region and a hydrophilic “head”region. The structure of the membrane bilayer is such that thehydrophobic (non polar) “tails” of the lipid monolayers orient towardsthe center of the bilayer while the hydrophilic “heads” orient towardthe aqueous phase.

In a liposome-drug delivery system, a hydrophilic therapeutic agent isentrapped in the aqueous phase of the liposome and then administered tothe patient. Alternatively, if the therapeutic agent is lipophilic, itmay associate with the lipid bilayer. Liposomes may be used to help“target” a drug to the active site or to solubilize hydrophobic drugsfor administration.

In an effort to increase the tumor selectivity of porphyrinphotosensitizers, the porphyrin compounds have been incorporated intounilamellar liposomes resulting in a larger accumulation and moreprolonged retention of the photosensitizer by both cultured malignantcells and experimental tumors in vivo. Jori et al., Br. J. Cancer,(1983) 48: 307–309; Cozzani et al., In Porphyrins in Tumor Phototherapy,Andreoni et al., eds., (1984) pp. 177–183, Plenum Press. The moreefficient targeting of tumor tissues by liposome-associated porphyrinsmay be partly due to the specific delivery of the phospholipid vesiclesto serum lipoproteins, which have been shown to interact preferentiallywith hyperproliferative tissue such as tumors through receptor mediatedendocytosis. In this manner the selectivity of porphyrin uptake bytumors is increased as compared with photosensitizers dissolved inaqueous solution. See Zhou et al., supra.

Accordingly, hematoporphyrin and hematoporphyrin dimethylester have beenformulated in unilamellar vesicles of dipalmitoyl-phosphatidyl choline(DPPC) and liposomes of dimyristoyl (DMPC) and distearoyl-phosphatidylcholine (DSPC). Zhou et al., supra; Ricchelli, F., New Directions inPhotodynamic Therapy, (1987) 847: 101–106; Milanesi, C., Int. J. Radiat.Biol., (1989) 55: 59–69. Similarly, HP, Photofrin® porfimer sodium, andtetrabenzo-porphyrins have been formulated in liposomes composed of eggphosphatidyl choline (EPC). Johnson, F. M. et., Proc. PhotodynamicTherapy: Mechanisms II, (1990), Proc. SPIE-Int. Soc. Opt. Eng., 1203:266–80.

Due to the importance of photodynamic therapy in the treatment ofcancer, there is a continuing need to identify new photosensitizerformulations that are stable, exhibit ease in manufacturing and whichselectively deliver the photosensitizer, particularly the morehydrophobic photosensitizers, to the target tissue in an efficientmanner.

SUMMARY OF THE INVENTION

The present invention involves a freeze dried pharmaceutical formulationcomprising a porphyrin photosensitizer, a disaccharide orpolysaccharide, and one or more phospholipids, which freeze-driedformulation forms liposomes containing a therapeutically effectiveamount of the porphyrin photosensitizer upon reconstitution with asuitable aqueous vehicle. The invention also relates to the liposomecomposition formed upon reconstitution with said aqueous vehicle.

The porphyrin photosensitizers usable in the practice of this inventioninclude any of the known porphyrin derivative compounds useful inphotodynamic therapy characterized in that they contain a porphyrin ringsystem. These include deuteroporphyrin, etioporphyrin, protoporphyrin,hematoporphyrin, pheophorbide and derivatives thereof. Particularlyuseful are hematoporphyrin and derivatives thereof as described in U.S.Pat. Nos. 4,649,151, 4,866,168, 4,889,129 and 4,922,934. The mostpreferred porphyrin photosensitizers usable in the present invention arethe (Gp) having a light absorption maximum between 670–780 nm whereinthe Gp is selected from the group consisting of those compounds havingthe formulae set forth in FIG. 1 and mixtures thereof and the metalatedand labeled forms thereof;

wherein each R¹ and R² is independently selected from the groupconsisting of carbalkoxy (2–6C), alkyl (1–6C) sulfonyl, aryl (6–10C)sulfonyl, aryl (6–10C); cyano; and —CONR⁵CO— wherein R⁵ is aryl (6–10C)or alkyl (1–6C);

each R³ is independently carboxyalkyl (2–6C) or a salt, amide, ester oracylhydrazone thereof, or is alkyl (1–6C); and

R⁴ is CHCH₂, CHOR^(4′), —CHO, —COOR^(4′), CH(OR^(4′))CH₃,CH(OR^(4′))CH₂OR^(4′), —CH(SR^(4′))CH₃, —CH(NR^(4′) ₂)CH₃, —CH(CN)CH₃,—CH(COOR^(4′))CH₃, —CH(OOCR^(4′))CH₃, —CH(halo)CH₃, or—CH(halo)CH₂(halo), wherein R^(4′), is H, alkyl (1–6C) optionallysubstituted with a hydrophilic substituent, or

wherein R⁴ is an organic group of <12C resulting from direct or indirectderivatization of vinyl, or

wherein R⁴ consists of 1–3 tetrapyrrole-type nuclei of the formula —L—Pwherein —L— is selected from the group consisting of

and P is selected from the group consisting of Gp which is of theformula 1–6 but lacking R⁴ and conjugated through the position shown asoccupied by R⁴ to L, and a porphyrin of the formula:

wherein each R is independently H or lower alkyl (1–4C);

wherein two of the bonds shown as unoccupied on adjacent rings arejoined to R³ and one of the remaining bonds shown as unoccupied isjoined to R⁴ and the other to L;

with the proviso that if R⁴ is CHCH₂, both R³ cannot be carbalkoxyethyl.

The preparation and use of such compounds is disclosed in U.S. Pat. Nos.4,920,143, and 4,883,790 hereby incorporated by reference. The mostpreferred compounds of the hydro-monobenzoporphyrins recited above arethe compounds of formulas 3 and 4 designated benzoporphyrin derivative(BPD) which have the formulas set forth in FIG. 2. These are hydrolyzedor partially hydrolyzed forms of the rearranged products of formula 3 or4, wherein one or both of the protected carboxyl groups of R³ arehydrolyzed. Particularly preferred is the compound referred to as BPD-MAin FIG. 2.

The liposomes of the present invention possess certain attributes whichmake them particularly suited for delivering the porphyrinphotosensitizers. Conventional liposomal formulations are preferentiallytaken up by the reticuloendothelial system (RES) organs such as theliver and spleen. When this occurs, the major portion of the liposomalencapsulated water insoluble drug is not available to tumor sites sinceit is localized in the RES. In contrast, the liposomes formed in thepresent invention are “fast breaking” in that the drug-liposomecombination is stable in vitro but when administered in vivo, thephotosensitizer is rapidly released into the bloodstream where itassociates with serum lipoproteins. It is believed that this inhibitsthe drug from being accumulated in non-target tissues such as the liver,where liposomes otherwise have a tendency to concentrate. The “fastbreaking” nature of the present liposomes may be due to the manner inwhich the porphyrin photosensitizer associates with the lipid bilayer ofthe liposomes of the present invention.

In addition, the particular combination of a disaccharide orpolysaccharide and one or more phospholipids forms a liposomalformulation which gives liposomes which exhibit excellentreproducibility in terms of particle size. Reproducibility and narrowparticle size distribution of the liposomal solution upon reconstitutionwith water is enhanced by an increased speed of hydration since a delayin hydration results in larger liposomes or precipitation of the drug.The addition of the disaccharide or polysaccharide providesinstantaneous hydration and the largest surface area for depositing athin film of the drug-phospholipid complex. This thin film provides forfaster hydration so that when the liposome is formed by adding theaqueous phase, the liposomes formed are of sufficiently small anduniform particle size such that the composition can be sterile filteredwithout any pre-filtering or separation of components with largerparticle size. This provides significant advantages in terms ofmanufacturing ease. In addition, the present liposomes provide 80–100%encapsulations of a drug which is expensive and requires a complicatedsynthetic procedure to produce. Thus, there is no reworking necessaryand very little waste of the drug.

Disaccharides or polysaccharides are preferred to monosaccharides. Tokeep the osmotic pressure of the liposome formulation similar to blood,no more than 4–5% monosaccharides can be added. In contrast, the sameosmotic pressure can be generated with 9–10% of a disaccharide. Thishigher amount of disaccharide provides for the larger surface area whichleads to the smaller particle size when the lyophilyzed liposomes arereconstituted.

The preferred liposomal formulation of the present invention forincorporation of porphyrin photosensitizers comprise a disaccharide orpolysaccharide, and one or more phospholipids which may be aphosphatidyl choline and a phosphatidyl glycerol. The disaccharide orpolysaccharide are preferably chosen from lactose, trehalose, maltose,maltrotriose, palatinose, lactulose or sucrose.

The preferable phospholipids are phosphatidyl cholines such asdimyristoyl phosphatidyl choline (DMPC), phosphatidyl choline (PC),dipalmitoylphosphatidy choline (DPPC), distearoylphosphatidyl choline(DSPC), soy phosphatidyl choline or egg phosphatidyl choline. Thepreferable phosphatidyl glycerols are dimyristoylphosphatidylglycerol(DMPG), and egg phosphatidylglycerol (EPG). Other phospholipids that maybe incorporated in the liposomes of the present invention arephosphatidyl ethanolamine, phosphatidic acid, phosphatidylserine andphosphatidylinositol. More preferably the liposomes comprise lactose,dimyristoylphosphatidyl choline (DMPC) and egg phosphatidylglycerol(EPG). The disaccharide or polysaccharide and phospholipid areformulated in a preferred ratio of about 10–20 to 0.5–6, respectively,most preferably 10 to 1.5–4.0.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects, features and advantages will occur from the followingdescriptions of preferred embodiments and the accompanying drawings inwhich;

FIG. 1 shows the structure of green porphyrin (Gp) compounds used in theliposomal formulations of the invention.

FIG. 2 shows the structure of four preferred forms of thehydro-monobenzoporphyrin derivatives of formulas 3 and 4 (BPDs).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a pharmaceutical liposome formulationof a porphyrin photosensitizer for use in the photodynamic therapy ordiagnosis of tumors, or for a variety of other therapeutic applications.The liposomes are formed upon addition of an aqueous vehicle to afreeze-dried formulation of a porphyrin photosensitizer, a disaccharideor polysaccharide, and one or more phospholipids such as phosphatidylcholines or phosphatidyl glycerols. The presence of the disaccharide orpolysaccharide in the formulation yields liposomes which have extremelysmall and narrow particle size, in which the porphyrin photosensitizersmay be stably incorporated into the liposome in an efficient manner withencapsulation efficiency approaching 80–100% of the drug. The liposomesexhibit physical and chemical stability such that they retainincorporated porphyrin drugs without leakage upon prolonged storage, aseither a reconstituted liposomal suspension or cryodesiccated powder.For example, BPD-MA, a preferred porphyrin photosensitizer, maintainedits potency in the cryodesiccated liposome formulation for a period ofat least nine months at room temperature and had a projected shelf lifeof at least two years.

The class of porphyrin photosensitizers preferably utilized in thepresent invention are the hydro-monobenzoporphyrins (GP) disclosed inU.S. Pat. Nos. 4,920,143 and 4,883,790, and most preferably thecompounds designated benzoporphyrin derivative (BPD), particularlyBPD-MA having the formula set forth in FIG. 2.

Liposomes containing a selected porphyrin photosensitizer as describedherein may be prepared by dissolving the porphyrin photosensitizer, thephospholipids and other optional adjuvants such as antioxidants inmethylene chloride or other suitable organic solvents. The resultingsolution is dried under vacuum until the organic solvent is evaporated.The solid residue is dispersed in an aqueous solution of thedisaccharide or polysaccharide and homogenized. The solution is thenfreeze dried for storage and reconstituted prior to administration witha suitable aqueous vehicle such as sterile water for injection. Uponreconstitution, liposomes are formed which incorporate a therapeuticallyeffective amount of the porphyrin photosensitizer.

The liposomal formulation of the present invention provides liposomes ofsufficiently small and narrow particle size such that it can bemanufactured without filtering to separate off larger particles orutilizing other mechanical methods of obtaining a narrow distribution ofparticle size.

As noted, the preferred phospholipids are the phosphatidyl cholines suchas dimyristoyl phosphatidyl choline (DMPC), phosphatidyl choline (PC),dipalmitoyl-phosphatidyl choline (DPPC) and distearoylphosphatidylcholine (DSPC) with DMPC being preferred. The preferred phosphatidylglycerols are dimyristoyl phosphatidylglycerol (DMPG) and eggphosphatidyl-glycerol (EPG) with DMPG being preferred. The preferreddisaccharides or polysaccharides are lactose, trehalose, maltose,maltotriose, palatinose, lactulose or sucrose with lactose or trehalosebeing most preferred. The disaccharide and phospholipids are formulatedin a preferred ratio of about 10–20 to 0.5–6 respectively, mostpreferably 10 to 1.5–4.0.

A preferable but not limiting formulation is lactose or trehalose,dimyristoyl phosphatidyl choline and egg phosphatidyl glycerol in aconcentration ratio of 10 to 0.94–1.88 to 0.65–1.30, respectively.

Other optional ingredients in the liposomal formulation are antioxidantssuch as butylated hydroxytoluene, α-tocopherol and ascorbyl palmitate.

The use of these porphyrin photosensitizers incorporated in liposomesfor the treatment or diagnosis of cancer is described herein as a neweffective treatment or therapeutic method. The liposomal formulationsare useful in sensitizing neoplastic cells or other abnormal tissueincluding infectious agents to destruction by exposure to light usingpreferably, visible light. Upon photoactivation, the porphyrinphotosensitizer promote the formation of singlet oxygen which isresponsible for the cytotoxic effect. In addition, the porphyrinphotosensitizers, when photoactivated, will fluoresce when subjected toappropriate excitation wavelengths. This fluorescence can be used tolocalize the tumor or other target tissue. By incorporating theporphyrin photosensitizer in the liposomes of the present invention,more efficient sensitization of tumor tissues can be obtained.

Generally speaking, the concentration of the porphyrin photosensitizerin the liposome depends upon the nature of the photosensitizer used.When the benzoporphyrin derivatives such as BPD-MA are used, thephotosensitizer is incorporated in the liposomes at a concentration ofabout 0.10% up to 0.5% w/v, yielding a reconstituted solution of up to5.0 mg/ml.

Such liposomes are typically administered parenterally. Injection may beintravenous, subcutaneous, intramuscular, intrathecal, or evenintraperitoneal. The liposomes could be administered by aerosolintranasaly or intrapulmonarily. The freeze dried powder may be packedin vials for reconstitution with sterile water prior to injection. Ofcourse, these compositions may also contain minor amounts of nontoxic,auxiliary substances such as pH buffering agents and the like.

The quantity of photosensitizer liposome formulations to be administereddepends on the choice of active ingredients, the conditions to betreated, the mode of administration, the individual subject and thejudgement of the practitioner. Generally speaking, dosages in the rangeof 0.05–10 mg/kg may be needed. The foregoing range is of course merelysuggestive, as the number of variables in regard to an individualtreatment regime is large and considerable excursions from theserecommended values are expected.

For use as a diagnostic in localizing tumor tissue or in localizingatherosclerotic plaques, the compounds or conjugates of the inventionare administered systemically in the same general manner as is knownwith respect to photodynamic therapy. The waiting period to allow thedrugs to clear from tissues to which they do not accumulate isapproximately the same, about 30 minutes to 10 hours. After thecompounds of the invention or their conjugates have been permitted tolocalize, the location of the target tissue is determined by detectingthe presence of the drug.

For diagnosis, the compounds incorporated in the liposomes may be usedalong with, or may be labeled with, a radioisotope or other detectingmeans. If this is the case, the detection means depends on the nature ofthe label. Scintigraphic labels such as technetium or indium can bedetermined using ex vivo scanners. Specific fluorescent labels can alsobe used, but these require prior irradiation, as does the detectionbased on fluorescence of the compounds of the invention themselves.

For activation of the photosensitizer of the invention, any suitableabsorption wavelength is used. This can be supplied using the variousmethods known to the art for mediating cytotoxicity or fluorescenceemission, such as visible radiation, including incandescent orfluorescent light sources or photodiodes, such as light emitting diodes.Laser light is also used for in situ delivery of light to the localizedphotosensitizer In a typical protocol, several hours before irradiation,approximately 0.5–1.5 mg/kg of the green porphyrin is injectedintravenously and then excited by an appropriate wavelength.

The methods of preparation of liposomal porphyrins of the presentinvention and photodynamic treatment therewith described in the Examplescontained later herein are readily adapted to the production and use ofanalogously described liposomes by simple substitutions of appropriateporphyrins, phospholipids or methods.

Either unilamellar or multilamellar or other types of liposomes may beused in the practice of the present invention. They may be prepared in asuspension form or may be formed upon reconstitution of a lyophilizedpowder containing the porphyrin-phospholipid-saccharide composition withan aqueous solution.

These following examples are presented to describe preferredembodiments, utilities and attributes of the present invention but arenot meant to limit the invention. For example, although DMPC and EPGwere used to form liposomes, these particular phospholipids are by nomeans the only available usable lipid forms known to those skilled inthe art. Nor do the particular methods of forming or preparing theliposomes described herein constitute the only methods for preparingliposomes contemplated by the present invention. Moreover, although theexamples imply the photosensitizer BPD-MA, the procedures, results andpreparations should be similar for other porphyrin photosensitizers.

EXAMPLE 1 Preparation of Liposomes Containing BPD-MA

BPD-MA was synthesized as described in U.S. Pat. Nos. 4,920,143 and4,883,790, incorporated herein by reference. Liposomes were preparedaccording to the following general procedure:

BPD-MA, butylated hydroxytoluene, ascorbyl palmitate and thephospholipids are dissolved in methylene chloride and the solution isfiltered through a 0.22 micron filter. The solution is then dried undervacuum using a rotary evaporator until the methylene chloride level inthe solid residue is not detectable by gas chromatography. A 10%lactose/water for injection solution is then prepared and filteredthrough a 0.22 micron filter. The lactose/water solution is warmed toabout 35° C. and added to the flask containing the solid residue of thephotosensitizer/phospholipid. The solid residue is dispersed in the 10%lactose/water solution and stirred for about one hour, cooled, andpassed through a homogenizer. The solution is then filtered through a0.22 micron Durapore® hydrophilic filter. Optionally, the solution mayfirst be prefiltered with a 5.0 micron prefilter. The filtrate iscollected, filled into vials and freeze dried and stored underrefrigeration. The freeze dried composition is reconstituted with waterfor injection prior to administration.

Using the foregoing procedure, several different preparations of theBPD-MA liposomal composition were prepared as follows:

EXAMPLE 1A

Preparation of a Liposomal Drug Delivery System Containing BPD-MA up to4.0 mg/ml Ingredient Amount % w/v BPD-MA 0.2 to 0.4 DimyristoylPhosphatidyl 0.94 to 1.88 Choline Egg Phosphatidyl 0.65 to 1.30 GlycerolLactose or  8.0 to 12.0 Trehalose Ascorbyl Palmitate 0.002 to 0.004Butylated Hydroxy 0.0002 to 0.0004 Toluene Water for Injection Q.S.

EXAMPLE 1B

Preparation of a Liposomal Drug Delivery System Containing BPD-MA up to3.0 mg/ml Ingredient Amount % w/v BPD-MA 0.2 to 0.3 DimyristoylPhosphatidyl 1.7 to 2.6 Choline Soy Phosphatidyl 2.3 to 3.5 CholineLactose or Trehalose  8.0 to 12.0 Ascorbyl Palmitate 0.002 to 0.003Butylated Hydroxy 0.0002 to 0.0003 Toluene Water for Injection Q.S.

EXAMPLE 1C

Preparation of a Liposomal Drug Delivery System Containing BPD-MA up to5.0 mg/ml Ingredient Amount % w/v BPD-MA 0.2 to 0.5 PhosphatidylEthanolamine 1.0 to 2.5 Egg Phosphatidyl  0.7 to 1.75 Choline Lactose orTrehalose  8.0 to 12.0 Ascorbyl Palmitate 0.002 to 0.005 ButylatedHydroxy 0.0002 to 0.0005 Toluene Water for Injection Q.S.

EXAMPLE 1D

Preparation of a Liposomal Drug Delivery System Containing BPD-MA up to4.0 mg/ml Ingredient Amount % w/v BPD-MA 0.2 to 0.4 DimyristoylPhosphatidyl 1.13 to 2.26 Choline Phosphatidic Acid 0.43 to 0.86 Lactoseor Trehalose  8.0 to 12.0 Ascorbyl Palmitate 0.002 to 0.004 ButylatedHydroxy 0.0002 to 0.0004 Toluene Water for Injection Q.S.

EXAMPLE 1E

Preparation of a Liposomal Drug Delivery System Containing BPD-MA up to1.0 mg/ml Ingredient Amount % w/v BPD-MA 0.1 Egg Phosphatidyl Choline0.55 Egg Phosphatidyl Glycerol 0.32 Lactose 8.0 to 12.0 AscorbylPalmitate 0.002 Butylated Hydroxy 0.0002 Toluene Water for InjectionQ.S.

EXAMPLE 1F

Ingredient Amount % w/v BPD-MA 0.2 to 0.3 Dimyristoyl Phosphatidyl 1.1to 1.6 Choline Phosphatidic Acid 0.4 to 0.7 Lactose or Trehalose  8.0 to12.0 Ascorbyl Palmitate 0.002 to 0.005 Butylated Hydroxy 0.0002 to0.0005 Toluene Water for Injection Q.S.

EXAMPLE 2 Physical and Chemical Stability of Liposomal BPD-MA

The physical stability of the liposomal BPD-MA was assessed bymonitoring the particle size distribution and osmolarity of thereconstituted solution over time when stored at various temperatures. Inall cases the mean particle size distribution was less than 200 nm.Osmolarity also showed no significant difference. The results are shownin Table 1. The data supports the physical stability of this dosageform.

TABLE I Stability of Liposomal BPD-MA for Injection 25 mg/vial MeanParticle Size Distribution Osmolarity nm mosm/kg Initial 170 295  3° C.-168 287 1 Month  3° C.- 157 291 3 Month  3° C.- 189 281 6 Month 23° C.-147 308 1 Month 23° C.- 155 291 3 Month 23° C.- 172 285 6 Month 30° C.-169 305 1 Month 30° C.- 134 291 3 Month 30° C.-75% RH 155 287 1 Month30° C.-75% RH 132 294 3 Month Light Cabinet 300 0.25 MonthThe chemical stability of the constituted dosage form was followed bymonitoring the potency, degradation products and pH of the reconstitutedsolution. The potency of the reconstituted parenteral dosage form wasassessed by chromatography with potency calculated on as is basis. Thepotency of the cryodesiccated powder showed a slight change from initialup to six month period at 3 or 23° C. with data ranging from 100.0–98.2percent of labeled claims. The results are shown in Table II.

TABLE II Stability of Liposomal BPD-MA for Injection 25 mg/vial PotencyAssay Potency Degradation BPD-MA Assay BPD-MA Product mg/vial 0/0 LabelHPLC Area 0/0 pH Initial 25.1 100.4 0.82 6.8  3° C.- 25.0 100.0 0.81 6.41 Month  3° C.- 25.1 100.4 0.72 6.4 3 Month  3° C.- 24.7 98.8 0.80 6.4 6Month 23° C.- 25.3 101.2 0.83 6.4 1 Month 23° C.- 25.0 100.0 0.85 6.4 3Month 23° C.- 24.7 98.6 0.80 6.2 6 Month 30° C.- 25.2 100.8 0.83 6.3 1Month 30° C.- 25.1 100.2 0.85 6.3 3 Month 30° C.-75% RH 25.2 100.8 0.806.3 1 Month 30° C.-75% RH 24.6 98.4 0.80 6.3 3 Month Light Cabinet 24.698.2 0.98 6.4 0.25 Month

EXAMPLE 3 Distribution of Liposomal BPD-MA in Human Blood

The liposomal BPD-MA of the present invention was incubated in humanblood for varying time periods and analyzed to determine thedistribution of the drug to various blood compartments.

Table III shows the comparison of the distribution of liposomal¹⁴C-BPD-MA (formulated) and ¹⁴C-BPD-MA in DMSO (non-formulated) betweenplasma and whole blood cells. ¹⁴C-BPD-MA at 25 μg/ml was incubated withwhole blood at 4° C.

Table IV shows the distribution of liposomal ¹⁴C-BPD-MA in plasma after1, 6 and 24 h incubation. Rudel's density gradient ultracentrifugationwas used to obtain fractions. Radioactivity is expressed as a percentageof total radioactivity in the plasma (mean±S.D.) n=2).

Table V shows the distribution of ¹⁴C-BPD-MA in DMSO (expressed as % oftotal radioactivity) between human plasma fractions obtained by Rudel'sdensity gradient centrifugation, following 1 and 24 h incubation. Eachvalue represents mean±S.D. (n=3).

The results shown in the following Tables III, IV and V demonstrate the“fast breaking” nature of the liposomal formulation of the presentinvention. As shown above the active drug associates rapidly with thelipoprotein compartment of the blood which in turn acts as a circulatingreservoir of the drug.

TABLE III % of Total Counts Plasma Red and White Blood Cells Time(h)Liposomal DMSO Solution Liposomal DMSO Solution 1 86.3 92.5 13.7  7.5 694.1 — 5.9 — 24 88.7 84.0 11.3 16.0

TABLE IV % of Total DPM Fraction Composition 1 h 6 h 24 h 1 + 2lipoproteins 91.1 ± 0.9  89.8 ± 0.9  90.4 ± 4.9  3 salt & water 1.8 ±0.5 2.5 ± 0.4 0.9 ± 0.2 4 albumin 6.6 ± 0.1 5.3 ± 2.3 6.5 ± 0.1 5 otherproteins 0.6 ± 0.2 2.6 ± 3.0 2.4 ± 2.2

TABLE V % Total DPM Fraction Composition 1 hour 24 hours 1 + 2lipoproteins 49.1 ± 2.6  86.7 ± 2.2  3 salt & water 13.1 ± 2.0  7.41 ±0.8  4 albumin 35.9 ± 0.1  4.9 ± 2.9 5 other plasma 1.8 ± 1.0 1.1 ± 0.1proteins

EXAMPLE 4 Antitumor Activity of Liposomal BPD-MA

Dose-response curves of liposomal benzoporphyrin derivative monoacid(BPD-MA) were obtained by exposing tumor-bearing mice treated withvarious doses of drug to 150 J/cm² of 690 nm laser light. The resultsindicated an ED₅₀ in the region of 1.5 mg/kg.

DBA/2 male mice carrying M1-S tumors were shaved and depilated at least24 hours prior to treatment. Liposomal BPD-MA was injected i.v., andafter a 3 hour waiting period, during which time the animals were keptin the dark, the tumor site was exposed to 150 J/cm² of 690 nm lightfrom an argon ion pumped dye laser. The animals were then returned tothe cage, and observed over the next 20 days.

Dose Groups Animals BPD-MA 0 mg/kg 2 × 10 BPD-MA 0.5 mg/kg 2 × 10 BPD-MA1.0 mg/kg 2 × 10 BPD-MA 1.5 mg/kg 2 × 10 BPD-MA 2.0 mg/kg 2 × 10

Two series of experiments were carried out, each consisting of 5 groupsof animals treated with 0, 0.5, 1.0, 1.5, or 2.0 mg/kg liposomal BPD-MA.(See Table VI)

100% of the animals at the 2 mg/kg dose point were tumor-free at day 7in both series I and II. By day 14, 30% of the tumors recurred in seriesI, and 20% in series II, and by day 20, 60% of the mice were tumorpositive in series I and 30% in series II.

At the 1.5 mg/kg point, 70% of the animals in series I and 80% in seriesII were tumor-free at Day 7, 30% in both series were tumor-free at Day14, and 10% and 20% were tumor-free at Day 20.

The 1.0 mg/kg dose points in the two series were dissimilar in that 40%of the animals were tumor-free at day 7 in series I, 20% at day 14, and10% at Day 20. In series II, 90% of the animals were tumor-free at day7, 40% at day 14 and 30% at day 20.

No effect was noted at either the 0.5 mg/kg or the 0 mg/kg dose points.The tumors continued to grow at the normal rate.

The disparity between the two series at the 1.0 mg/kg dose point makesit difficult to determine an ED₅₀. However, at this time we can deducethat the ED₅₀ will lie in the region of 1.5 mg/kg.

The following Table VI provides the number of animals remaining tumorfree at Day 7, 14 and 20 after treatment with varying doses of liposomalBPD-MA and 150 J/cm² 690 nm laser light.

TABLE VI Drug Dose # Animal D7 D14 D20 Series I 0.0 mg/kg 10 0 — — 0.5mg/kg 10 0 — — 1.0 mg/kg 10 4 2 1 1.5 mg/kg 10 7 3 1 2.0 mg/kg 10 10 7 4Series II 0.0 mg/kg 10 0 — — 0.5 mg/kg 10 0 — — 1.0 mg/kg 10 9 4 3 1.5mg/kg 10 8 3 2 2.0 mg/kg 10 10 8 7

1. A liposomal formulation comprising liposomes that comprisephosphatidyl choline, phosphatidyl glycerol and a porphyrin macrocyclephotosensitizer, wherein said liposomes have a mean particle sizedistribution of less than 200 nm.
 2. The liposomal formulation of claim1 in freeze-dried form.
 3. The liposomal formulation of claim 1, whereinsaid sugars are selected from disaccharides or polysaccharides.
 4. Theliposomal formulation of claim 3 wherein said disaccharides are selectedfrom lactose or trehalose.
 5. The liposomal formulation of claim 1wherein the lipid bilayer of said liposomes consists essentially ofdimyristoyl phosphatidyl choline and egg phosphatidyl glycerol.
 6. Theliposomal formulation of claim 1 wherein said porphyrin macrocyclephotosensitizer is a hydro-monobenzoporphyrin (Gp) of any one of thefollowing formulas

and having a light absorption maximum between 670–780 nm, mixturesthereof, and the metallated and labeled forms thereof, wherein each R¹and R² is independently selected from the group consisting of carbalkoxy(2–6C), alkyl (1–6C) sulfonyl, aryl (6–10C) sulfonyl, aryl (6–10C);cyano; and —CONR⁵CO— wherein R⁵ is aryl (6–10C) or alkyl (1–6C); each R³is independently carboxyalkyl (2–6C) or a salt, amide, ester oracylhydrazone thereof, or is alkyl (1–6C); and R⁴ is —CH═CH₂,—CHOR^(4′), —CHO, —COOR^(4′), —CH(OR^(4′))CH₃, —CH(OR^(4′))CH₂OR^(4′),—CH(SR^(4′))CH₃, —CH(NR^(4 ′) ₂)CH₃, —CH(CN)CH₃, —CH(COOR^(4′))CH₃,—CH(OOCR^(4′))CH₃, —CH(halo)CH₃, or —CH(halo)CH₂(halo), wherein R^(4′)is H, alkyl (1–6C) optionally substituted with a hydrophilicsubstituent, an organic group of less than 12C resulting from direct orindirect derivatization of vinyl, or 1–3 tetrapyrrole-type nuclei of theformula —L—P wherein —L— is selected from the group consisting of:

and P is selected from the group consisting of Gp which is of theformula of FIG. 1–2, but lacking R₄ and conjugated through the positionshown as occupied by R⁴ to L; with the proviso that, if R⁴ is —CH═CH₂,both R³ groups cannot be carbalkoxyethyl.
 7. The liposomal formulationof claim 6 wherein each R³ is —CH₂CH₂COOH or salt, amide, ester oracylhydrazone thereof.
 8. The liposomal formulation of claim 6 whereineach of R¹ and R² is carbalkoxy (2–6C).
 9. The liposomal formulation ofclaim 7 wherein each of R¹ and R² is carbalkoxy (2–6C).
 10. Theliposomal formulation of claim 6 wherein said hydro-monobenzoporphyrin(Gp) is selected from the group consisting of: BPD-DA wherein R¹ and R²thereof are carbomethoxy; BPD-DB wherein R¹ and R² thereof arecarbomethoxy; BPD-MA wherein R¹ and R² thereof are carbomethoxy and R ismethyl; and BPD-MB wherein R¹ and R² thereof are carbomethoxy and R ismethyl.
 11. The liposomal formulation of claim 10 wherein saidhydro-monobenzoporphyrin (Gp) is BPD-MA wherein R¹ and R² thereof arecarbomethoxy and R is methyl.
 12. The liposomal formulation of claim 5wherein the amounts of photosensitizer, dimyristoyl phosphatidylcholine, and egg phosphatidyl glycerol in said liposomes are, relativeto each other on a per weight basis, about 0.2 to 0.4 of porphyrin; 0.94to 1.88 of dimyristoyl phosphatidyl choline; and 0.65 to 1.30 of eggphosphatidyl glycerol.
 13. The liposomal formulation of claim 12 whereinthe amount of sugar, relative to said amounts of photosensitizer,dimyristoyl phosphatidyl choline, and egg phosphatidyl glycerol in saidliposomes on a per weight basis, is about 8.0 to 12.0 of sugar when saidsugar is a disaccharide, or about half that amount if said sugar is amonosaccharide.
 14. The liposomal formulation of claim 5 furthercomprising an antioxidant.
 15. The liposomal formulation of claim 14wherein said antioxidant is butylated hydroxytoluene or L-ascorbic acid6-palmitate.
 16. The liposomal formulation of claim 1 further comprisinga pharmaceutically acceptable excipient.
 17. A method of providingphotodynamic therapy to a subject comprising administering a formulationaccording to claim 1 to said subject wherein the porphyrin macrocyclephotosensitizer, after release from said formulation, is capable oflocalizing to target tissues or cells, and irradiating said tissues orcells at an appropriate wavelength of light after passage of sufficienttime for said porphyrin macrocycle photosensitizer to localize.
 18. Amethod of providing photodynamic therapy to a subject comprisingadministering a formulation according to claim 5 to said subject whereinthe porphyrin macrocycle photosensitizer, after release from saidformulation, is capable of localizing to target tissues or cells, andirradiating said tissues or cells at an appropriate wavelength of lightafter passage of sufficient time for said porphyrin macrocyclephotosensitizer to localize.
 19. The liposomal formulation of claim 1wherein the ratio of sugar to phospholipid is about 10–20 to 0.5–6. 20.The liposomal formulation of claim 19 wherein the ratio of sugar tophospholipid is 10 to 1.5–4.0.
 21. The liposomal formulation of claim 1,wherein said liposomes are fast breaking and rapidly release thephotosensitizer into the bloodstream to associate with lipoproteins uponin vivo administration.
 22. The liposomal formulation of claim 1,wherein the osmolarity of said liposomes is that of human blood.
 23. Apharmaceutical composition comprising the liposomal formulation ofclaim
 1. 24. The liposomal formulation of claim 1, wherein saidphosphatidyl choline is dimyristoyl phosphatidyl choline.
 25. Theliposomal formulation of claim 1, wherein said phosphatidyl glycerol isegg phosphatidyl glycerol.
 26. The liposomal formulation of claim 1,wherein said liposomes further comprise one or more sugars.